Present study demonstrated that fibrillar β-amyloid peptide (fAβ 1-42 ) induced ATP release, which in turn activated NADPH oxidase via the P2X 7 receptor (P2X 7 R). Reactive oxygen species (ROS) production in fAβ1-42-treated microglia appeared to require Ca 2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca 2+. Considering previous observation of superoxide generation by Ca 2+ influx through P2X 7 R in microglia, we hypothesized that ROS production in fAβ-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAβ1-42-induced Ca 2+ influx was mediated through P2X 7 R activation. In serial experiments, we found that microglial pretreatment with the P2X 7 R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 µM) or oxidized ATP (100 µM) inhibited fAβ-induced Ca 2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAβ1-42-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAβ-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.

Recent studies have reported that the "cholinergic anti-inflammatory pathway" regulates peripheral inflammatory responses via α 7 nicotinic acetylcholine receptors (α 7 nAChRs) and that acetylcholine and nicotine regulate the expression of proinflammatory mediators such as TNF-α and prostaglandin E 2 in microglial cultures. In a previous study we showed that ATP released by β -amyloid-stimulated microglia induced reactive oxygen species (ROS) production, in a process involving the P2X7 receptor (P2X7R), in an autocrine fashion. These observations led us to investigate whether stimulation by nicotine could regulate fibrillar β amyloid peptide (1-42) (fAβ 1-42)-induced ROS production by modulating ATP efflux-mediated Ca 2+ influx through P2X 7 R. Nicotine inhibited ROS generation in fAβ 1-42-stimulated microglial cells, and this inhibition was blocked by mecamylamine, a non-selective nAChR antagonist, and α -bungarotoxin, a selective α 7 nAChR antagonist. Nicotine inhibited NADPH oxidase activation and completely blocked Ca 2+ influx in fAβ 1-42 -stimulated microglia. Moreover, ATP release from fAβ 1-42-stimulated microglia was significantly suppressed by nicotine treatment. In contrast, nicotine did not inhibit 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP)-induced Ca 2+ influx, but inhibited ROS generation in BzATP-stimulated microglia, indicating an inhibitory effect of nicotine on a signaling process downstream of P2X7R. Taken together, these results suggest that the inhibitory effect of nicotine on ROS production in fAβ 1-42 -stimulated microglia is mediated by indirect blockage of ATP release and by directly altering the signaling process downstream from P2X 7 R.

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 µM) and bell-shaped. The concentrations of ATP, ATP-γS, ADP, and ADP-β S that showed maximal IL-10 release were 100, 10, 100, and 100 µM respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP ＞ 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), α,β-methylene ATP (α,β-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca 2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down-regulated by either MRS2179 (a P2Y 1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y 1 and P2Y 11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-γS (100 µM) was restored by TNP-ATP (an antagonist of the P2X 1 , P2X 3 , and P2X 4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y 12 receptor antagonist) or pertussis toxin (PTX; a G i protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y 11 receptor or the P2Y 1 receptor, respectively.